Introduction
The efficiency of sheep production is conditioned by fertility. Therefore,
detection of genetic variation in major genes which affect prolificacy traits
is an important prerequisite to improve the quantity, quality, and diversity
of production in the livestock industry (Notter, 1999). Antral follicle
count, ovulation rate, and litter size can be regulated by a set of major
genes that mainly belong to the bone morphogenetic protein (BMP) signalling pathway
(Hanrahan et al., 2004; Davis, 2005; Fabre et al., 2006; Pelosi et al.,
2015). These fecundity genes include bone morphogenetic protein receptor
1β (BMPR1b or FecB), growth and differentiation
factor 9 (GDF9 or FecG), and bone morphogenetic protein
(BMP15 or FecX), which encode the most important growth
factors involved in folliculogenesis (Mermillod et al., 2008). BMP15
is involved in the development of ovarian follicles and is a key regulator of
some processes in the granulosa cells, including cellular proliferation,
apoptosis prevention, and steroidogenesis (Juengel and McNatty, 2005).
The location and effect of various mutations in ovine
BMP15 mRNA
(ENSOARG00000009372) on ewe prolificacy. Mutations in the gray box are the
effect on the ewe litter size. Mutations in the black box have not been
documented to have an effect on the ewe litter size. The T755C mutation, the
green box, is the novel mutation that has been detected in Iranian sheep
breeds of this study.
In sheep, the BMP15 gene is located on chromosome X and encompasses
two exons, which are separated via a 5.4 kb intron. This gene has a 1179 base pair
open reading frame (ORF)
encoding an
immature polypeptide with 393 amino acid residues and an active mature
peptide that is 125 amino acids long (Galloway et al., 2000). As
reviewed in Fig. 1, 15 mutations have heretofore been identified in the sheep
BMP15 gene. Among them, six mutations of FecXI, FecXH, FecXB, FecXG,
FecXL, and FecXR increase ovulation rate and litter size in heterozygous
animals, while completely sterilizing in homozygous mutants (Galloway et al.,
2000; Hanrahan et al., 2004; Bodin et al., 2007; Martinez-Royo et al., 2008;
Monteagudo et al., 2009). In contrast, homozygous ewes for two mutations of
FecXGr and FecX0 are unexpectedly hyper-prolific and hence regarded as
striking variants in the sheep BMP15 gene (Demars et al., 2013). The
other six mutations, including the CTT deletion in signal peptide (B1),
g.200G > A, g.288G > A, and g.747T > C
(B3) in the proprotein region, as well as g.1037G > A and
g.1047G > A in the mature protein, have been reported as
ineffective variants. With the recent progress in the genetics field, there
is considerable evidence that the presence of silent mutations (such as B3,
g.288G > A, and g.1047G > A) can alter the protein
functionality (Parmley and Hurst, 2007; Sauna et al., 2007; Hunt et al.,
2014) and the phenotype variation through splicing accuracy, translation
loyalty, mRNA structure, and miRNA binding (Chamary and Hurst, 2005; Sauna
and Kimchi-Sarfaty, 2011; Cuevas et al., 2011). However, there is no
available data to attest to or controvert this approach for litter size
trait. Conversely, although numerous mutations have been detected in major
genes relevant to prolificacy of various sheep breeds around the world, the
Lleyn breed and many other highly prolific ewe breeds with triplet-birth
records, did not carry these known significant mutations (Mullen et al.,
2013). Therefore, screening highly prolific ewes for the known genes and
other candidate genes is essential to fulfill our knowledge about these
intra- and interbreed genetic variations.
Distribution of genotyped samples from ewes and rams of four Iranian
sheep breeds.
Breed
Ewe
Ram
Sum
Infertile
Singleton
Doubleton
Tripleton
Shal
1
13
19
0
16
49
Ghezel
–
20
30
–
7
57
Afshari
–
38
5
–
12
55
Lori-Bakhtiari
1
34
49
3
9
96
Total number
2
105
103
3
44
257
Primers and PCR conditions utilized for amplification of
exon 1 and exon 2 of the sheep BMP15 gene.
Primer sequence
Position
Amplicon
Annealing
Extension
(5′ → 3′)
size
temperature
temperature
(∘C/S)
(∘C/S)
F1: TCCTTGCCCTATCCTTTGTG
Exon 1
482
58.5/30
72/45
R1: CCCTCCCACCAGAACAATA
F2: GAAGCTAACGCTTTGCTCTTG
Exon 2
468
58/30
72/40
R2: GCCTTTAGGGAGAGGTTTGG
F3: GGCACTTCATCATTGGACAC
Exon 2
539
59/35
72/50
R3: CTGAGCTAGCTGCACCTTTG
Over the last decade, several studies have been conducted on screening
mutations in the BMP15 gene of Iranian sheep breeds (Barzegari et al.,
2010; Hafezian, 2011; Javanmard et al., 2011; Eghbalsaied et al., 2012; Zamani
et al., 2015; Doran et al., 2016; Ahmadi et al., 2016;Nadri et al.,
2016; Abdoli et al., 2017). Although novel mutations have been detected in
BMP15 in some Iranian sheep breeds (Asghari et al., 2009; Javanmard et
al., 2011; Zamani et al., 2015; Ahmadi et al., 2016; Nadri et al., 2016; Abdoli
et al., 2017), these mutations have either been non-significant or had a
partial effect on litter size in these breeds. Therefore,
responsible mutations in BMP15 have yet to be discovered in these breeds.
Thus, the aim of the present study was to screen the BMP15 gene in a group of
infertile, twin-birth, and triplet-birth ewes and to evaluate the mutation
effect on the litter size of Iranian Shal, Afshari, Ghezel, and
Lori-Bakhtiari breeds. These breeds are the most prominent native sheep in
northern, central, and southern parts of the Zagros mountain chain and have a
large carcass and fat-tail size (Eskandarinasab et al., 2010; Vatankhah
et al., 2016).
Materials and methods
Animals and sample collection
All the following procedures which were carried out on animals were approved
by the Animal Welfare Committee of Isfahan (Khorasgan) branch of the Islamic
Azad University. The total genotyped samples were comprised of 257 sheep with
49 Shal, 57 Ghezel, 55 Afshari, and 96 Lori-Bakhtiari sheep (Table 1). In
this study, blood samples of Shal, Ghezel, Afshari, and Lori-Bakhtiari sheep
breeds were obtained from the Bouinzahra region (Qazvin Province, situated at
36∘16′ N and 50∘00′ E); Miyandoaab Research Center and
the Simineh (Bukan) region (West Azerbaijan Province, located at
36.92∘ N and 46.16∘ E);
Khatoon Abad region (Isfahan
Province, located at 33.27∘ N and 52.36∘ E); and the three
regions of Shahr-e Kord, Lordegan, and Farsan (Chaharmahal and Bakhtiari
Province, situated at 32.32∘ N and 50.85∘ E), respectively.
Allelic and genotypic frequencies of the T750C mutation in
Iranian Afshari, Ghezel, Lori-Bakhtiari, and Shal sheep breeds.
Breed
Sex
Genotypic
Allelic
PIC
frequency
frequency
CC
CT
TT
C
T
Afshari
Ewe
0.18
0.26
0.56
0.31
0.69
0.34
Ram
0.25
0.33
0.42
0.42
0.58
0.37
Overall
0.20
0.28
0.52
0.34
0.66
0.35
Ghezel
Ewe
0.15
0.25
0.60
0.28
0.72
0.32
Ram
0.13
0.25
0.62
0.25
0.75
0.30
Overall
0.15
0.25
0.60
0.27
0.73
0.32
Lori-Bakhtiari
Ewe
0.10
0.13
0.77
0.16
0.84
0.23
Ram
0.0
0.5
0.5
0.25
0.75
0.30
Overall
0.10
0.15
0.76
0.17
0.83
0.23
Shal
Ewe
0.20
0.20
0.60
0.30
0.70
0.33
Ram
0.14
0.43
0.43
0.36
0.64
0.35
Overall
0.19
0.26
0.56
0.32
0.68
0.34
Total
–
0.14
0.22
0.64
0.25
0.75
0.30
DNA extraction and PCR amplification
Genomic DNA was extracted from the whole blood by the phenol–chloroform
method (Sambrook and Russell, 2006). Final DNA pellet was dissolved in the
double distilled water and was stored at -20 ∘C for further
experiments. Exon 1 and exon 2 of BMP15 were amplified by three
specific primers which were designed using the Primer3 software from the NCBI
website (Table 2). Polymerase chain reaction (PCR) was conducted in
25 µL total volume containing approximately 50–100 ng of genomic
DNA, 2.5 mM of MgCl2, 10 × PCR buffer (500 mM of KCl and Tris HCl
(pH 8.4)), 10 µM of forward–reverse primer, 0.2 mM of dNTPs, and
1 IU
Smart Taq DNA polymerase (catalogue number: TA8108C, SinaClon,
Iran). The PCR amplifications were carried out in a thermal cycler (Bio-Rad,
USA) by using a specific annealing temperature (Table 2).
SSCP analysis
The genotypic patterns of PCR products were determined using the single-strand conformation polymorphism (SSCP) method
with slight modifications (Orita et al., 1989). Briefly, a constant volume of
2 µL PCR products of each sample was mixed with 8 µL of gel
loading dye solution (98 % formamide, 10 mM EDTA, 0.025 %
bromophenol blue, and 0.025 % xylene cyanol). The mixture was vortexed and
denatured at 95 ∘C for 10 min and rapidly chilled on ice and loaded on
10 % polyacrylamide gels (acrylamide : bisacrylamide = 37.5 : 1).
Electrophoresis was carried out at 250 V for 24 h at 4 ∘C in 1 × TBE
buffer. Then, the DNA bands were visualized using silver staining (Bassam et
al., 1991). After the SSCP analysis, three samples from each distinctive
pattern were sequenced using both the forward and reverse primers (Bioneer,
Korea). The sequences were analysed using the ClustalW subprogram of BioEdit
software to execute the multiple sequence alignments (Hall et al., 2011).
Sequence and statistical analysis
The genotype and allele frequency, as well as Hardy–Weinberg equilibrium
test, were estimated using the GenAlEx 6.5 program (Peakall and Smouse, 2006).
Polymorphism information content (PIC) values were computed using HET software
version 1.8 (Ott, 2001). The GENMOD procedure in the SAS software was
implemented to analyse the association between genetic variation in the
BMP15
gene and the litter size records (Institute, 1985). Also, mean comparison
between different genotypes was carried out using the LSM (least-square mean)
option. A P value ≤ 0.05 was considered as the statistically
significant level.
Discussion
It has been well documented that heterozygote carriers for BMP15
mutations display an increased ovulation rate and litter size compared to the
wild-type ewes (Galloway et al., 2000; Hanrahan et al., 2004; Bodin et al.,
2007; Martinez-Royo et al., 2008; Monteagudo et al., 2009). The majority of
these mutations were located in the propeptide region of BMP15 or
GDF9 (Di Pasquale et al., 2004; Inagaki and Shimasaki, 2010; Abdoli
et al., 2016). The propeptide region of the protein is one of the most
important attributes affecting the post-translational processing of the
transforming growth factor beta (TGF β) superfamily (Liao et
al., 2003; Shimasaki et al., 2004; Di Pasquale et al., 2004).
In the current study, we screened BMP15 in a group of ewes with
single-, twin-, and triplet-birth lambing and a sample of rams as flock sires
in the four main breeds Lori-Bakhtiari, Shal, Ghezel, and Afshari. One SNP
was detected with PCR–SSCP and verified by sequencing of the amplicons.
Except for the GWAS data, this is the first report on the presence and
importance of this SNP (T755C) for ewe's litter size (rs55628000). From 20
genotyped samples of Iranian Ovis aries sheep, the frequency of
5 % was reported for the T755C mutation using next-generation
whole-genome sequencing. However, the current paper is the first report which
provides the population and association study on this mutation. The frequency
of this mutation varied from 0.17 to 0.34 in Lori-Bakhtiari and Afshari
breeds, respectively. Lack of Hardy–Weinberg equilibrium in this study
implied that natural or artificial selection favoured the mutated allele in
all four breeds. The majority of sheep breeds in Iran are kept under the
traditional animal husbandry system in rustic and nomadic production systems.
Thus, this unequal distribution indicates that the T755C mutation can be
associated with fitness traits which form the main practical index in the
traditional breeding systems of sheep species. Also, the PIC index indicated
the presence of a moderate genetic diversity for this mutation, which makes
it plausible for being included in the breeding programs (Botstein et al.,
1980). In our study, the ewes with the CT genotype had more lambs than those
with homozygous genotypes. This indicates that the mutation has a dominance
effect on the ewe litter size. It might be noteworthy that the infertile ewes
were also heterozygous for this mutation. Conversely, all ewes with
triplet-birth phenotypes were heterozygous carriers for this locus. These
unexpected results strongly suggested synergistic effects of the T755C
mutation with other mutation(s), which may induce over-dominance and
sterility when heterozygous and homozygous, respectively. Therefore, other
mutations in the non-sequenced region of BMP15 or other
above-mentioned genes may act through a nonadditive effect with the T755C
mutation and induce infertility or higher prolificacy. Similar to some of the
FecX variants that have been detected, the prolific ewes with the
heterozygote T755C genotype had an increased litter size, while no sterility
phenotype was detected in homozygous animals. However, no significant
differences between heterozygous and homozygous mutant types were observed in
the Lori-Bakhtiari or Afshari sheep breeds. The reason for these interbreed
differences can be related to the difference in the genetic background of
these breeds (Eghbalsaied et al., 2017). Our recent study on GDF9
mutations confirmed the high variation in the frequency of G1 and G4 SNPs in
these four evaluated breeds (Eghbalsaied et al., 2017). Sequencing of
GDF9 did not show any significant mutation in these infertile and
triplet-birth ewes (Eghbalsaied et al., 2017) as well as ewes with high
antral follicle count (Eghbalsaied et al., 2012). This may suggest the
presence of the T755C mutation with a different set of SNPs in other
candidate genes, such as AMPK (Foroughinia et al., 2017),
Cyp19 (Foroughinia et al., 2017), or leptin (Juengel et
al., 2015) in different sheep breeds.